3012
J . Am. Chem. SOC.1993,115, 30 12-301 3
Active-Site Electronic Structure Contributions to Electron-Transfer Pathways in Rubredoxin and Plastocyanin: Direct versus Superexchange Michael D. Lowery, Jeffrey A. Guckert, Matthew S.Gebhard, and Edward I. Solomon’
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Department of Chemistry Stanford University Stanford, California 94305 Received October 26, 1992
Plastocyanin (Pc), a blue copper protein,1-2and rubredoxin (Rd), a single iron-sulfur protein,, are important biological electron-transfer (ET) agents. In this study, we have used selfconsistent-field X a scattered-wave (SCF-Xa-SW) molecular orbital calculations, calibrated to charge-transfer (CT) absorption data, to determine the electronic contributions of the Pc and Rd active sites to ET pathways. Though much attention has been given to the long-range mediation of protein ET, this work focuses on the potentially important role of active-site metal-ligand covalency in the determination of effective ET pathways. In both proteins the redox active orbital is dominated by metalcysteine *-bonding interactions. The C T spectra of Pc and Rd, however, reveal a striking difference in the extent of *-bonding covalency. In the Pc active site, the HOMO is dominated by an exceptionally strong covalent CuS(Cys)p* bond which activates thecysteine (Cys) residue for efficient long-range ET. In the Rd active site, FeS(Cys)p* bonding in the HOMO is found to be extremely weak so that the rapid electron-self-exchange (ese) rate4 (-lo9 M-I SKI) is not consistent with an extended superexchange pathway. Therefore, a direct ET mechanism involving overlap with the FeS4 active site is predicted. Electronic structure calculations were performed using the QCPE releaseS of the SCF-Xa-SW code.6 The Pc site was modeled in C, symmetry by [Cu(SCH,)(C,H,N2)2(S(CH3)2)]+, and the Rd active site was modeled in D2d symmetry by [Fe(SCH3),,] I-. Sphere radii7J were optimized by reproducing the experimental CT spectra. In Pc, the trigonally distorted tetrahedral blue copper active site is buried -6 A from the protein surface and consists of a copper atom strongly coordinated to three nearly coplanar atoms (Na, of histidines (His) 37 and 87 and S, of Cys 84) and has an additional weak bond to Sa of methionine (Met) 92.9 Two ET pathways have been reported for P c . ~The shorter “adjacent” path (-6 A) proceeds from the Cu atom through the surfaceexposed His 87 residue and is involved in ese reactions and E T with anionic redox partners. The longer “remote” path (- 13 A) involves the Cys 84 and tyrosine (Tyr) 83 residues. Tyr 83 lies on the protein surface in a negatively charged region and favors ET reactions with cationic redox partners. A similar (Cys-His) ET pathway is present in the multicopper oxidases and in nitrite reductase.’ Author to whom correspondence should be addressed. ( I ) Solomon, E. 1.; Baldwin, M. J.; Lowery, M. D. Chem. Reo. 1992, 92, 521-542. (2) Adman, E. T. In Advances in frorein Chemistry; Anfinsen, C. B., Richards, F. M., Edsall, J. T., Eisenberg, D. S.. Eds.; Academic Press, Inc.: San Diego, CA, 1991; Vol. 42, pp 145-198. (3) IronSuljurfroreins;Lovenberg, W., Ed.; Academic Press: New York, 1973; Vol. I . (4) Jacks, C. A.; Bennett, L. E.; Raymond, W. N.; Lovenberg, W. f r o c . Nail. Acad. Sci. U.S.A. 1974, 71, 1 118-1 122. ( 5 ) Cook, M.; Case, D. A. Q C f E Program 465 1991, 23, 21-22. (6) Connolly, J. W . D. In Modern Theoretical Chemistry; Segal, G . A,. Ed.; Plenum: New York, 1977; Vol. 7, pp 105-132. (7) Gewirth, A. A.; Solomon, E. 1. J. Am. Chem. SOC.1988,110, 381 13819. (8) Bair, R. A.; Goddard, W. A. I . J. Am. Chem. SOC.1978, 100, 56695676. (9) Guss, J. M.; Freeman, H. C. J. Am. Chem. SOC.1983, 169, 521-563.
I 3oo00
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2oooo 15000 loo00 Energy (an-’) Figure 1. Electronic absorption spectra of (A) plastocyanin’ and
(e)
rubredoxin.’
Both pathways in Pc exhibit comparable ET rates of 104-103 M-I s - ~ . ~ OHowever, for tunneling through a structureless onedimensional barrier, the rate of E T (kET) is expected to decay exponentially with the distance ( ~ D Abetween ) the donor (D) and acceptor (A) sites and can be expressed as:11-13 k,T
= A’eXp(-@DA)(FC)
= (2*h)lT~~l’(Fc)
(1)
where TDAis the electronic tunneling matrix element connecting the donor and acceptor states and (FC) is the Franck-Condon factor. Using eq 1 with rDA = 6 A for the adjacent path and 13 A for the remote path,9 kET is calculated to be 1500X more rapid through theadjacent path. Beratan hasdevelopeda pathway model which estimates TDAas a function of the number of bonds and contacts connecting the donor and acceptor sites.” For Pc, considering only covalent linkages between Cu-His(87) (4 bonds) and Cu-Cys(84)-Tyr(83) (12bonds), the pathway model predicts that the adjacent path is -3OOOX more rapid for ET. The observed ET rates, however, do not show this great disparity. The apparent near equality of kET for the two E T paths can be related to the anisotropic covalency of the HOMO as outlined below. In the electronic absorption spectrum of Pc, Figure lA, the dominant CT band at 598 nm (16 700 cm-I) derives from a S(Cys)p* Cu2+transition.’ Normally, u C T transitions are most intense, as is observed in Rd and other metal-thiolate systems.I4 The reversed CT intensity pattern results from the strong Cu-S(Cys) bond (2.13A9) whichorientsthehalfsccupied Cu dxz-yzorbital so that it is bisected by the CuS(Cys) bond and significantly overlaps the S(Cys)px orbital. Using SCF-XuS W calculations expanded to include imidizole ligation, the HOMO wave function (*HOMO) character is found to be 42% Cu dxz-yzr46% S(Cys)p*, 2.3% N(His) (1.2% each), and -0.0% S(Met), in excellent agreement with experimental results from Cu L-edge15 and S K-edgel6 X-ray absorption spectroscopies. The dominant S(Cys)pr character ( y ~ ’= 0.46, where y~ is the ligand coefficient in *HOMO) activates the Cys ligand for E T as
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(IO) Sykes, A. G . Srruct. Bonding 1991, 75, 177-224. ( 1 I ) Beratan, D. N.; Betts, J. N.; Onuchic. J. N. Science 1991,252, 12851288. (12) Newton, M. D. Chem. Reo. 1991, 91, 767-792. ( 1 3) Marcus, R. A,; Sutin, N. Eiochim. Eiophys. Acta 1985, 81 I, 265322. (14) Solomon, E. 1.; Lowery, M.D.; LaCroix, L. B.; Root, D. R. Merhods
Enzymol., in press. (15) George, S. J.; Lowery, M.D.; Solomon, E. 1.; Cramer. S. P. J . Am. Chem. Soc., in press. (16) Shadle, S. E.; Penner-Hahn, J. E.; Schugar, H. J.; Hedman, B.; Hodgson, K. 0.;Solomon, E. I. J. Am. Chem. SOC.1993, 115, 767-776.
OOO2-7863/93/ 1515-3012$04.O0/0 0 1993 American Chemical Society
Communications to the Editor
J . Am. Chem. SOC.,Vol. 115, No. 7, 1993 3013
*q
w> cy541
T y r 83
NSI His :
1.2 8
cvs 39
Figure 3. *HOMO wave function contour plot superimposed on the R d structure. (A) Polypeptide backbone for R d using the 4rxn coordinatesIg deposited in the Brookhaven protein data base.2i Side groups of Cys 6, 9,39, and 42 a r e included. (B) Expanded view of the proposed E T path (rotated 90' from A) with qHOMosuperimpOsed onto the crystal structure (only S, and Cb of Cys 39 a r e shown for clarity). Contour values a r e the same as those in Figure 2.
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value, indicating 35% ligand character in du. The intensity of Figure 2. ql{oMowave function contour plot superimposed on the Pc the S p r Fedr C T transitions is only 3% that of the Spu structure. (A) Polypeptide backbone for Pc using the lpcy coordinates9 Fedu transitions. As the intensity of a CT absorption band is deposited in the Brookhaven protein data base.2s Side groups of His 37, directly proportional to the extent of ligand mixing in the accepting His 87, Cys 84, and T y r 83 a r e included. (B) Expanded view of the metal orbital,22the total S p r character in the Fedr orbitals is proposedsuperexchange paths with P ~ o ~ o s u p e r i m p o sontothecrystal ed structure (viewed rotated 180' around the Cu-S (Cys) bond from A). 1%. Moreover, this value represents the maximum amount of Contour values a r e a t f0.16, fO.08, f0.04,*0.02, and fO.O1 ( e / ~ ( ~ ~ ) l / ~ ,S p r mixing in the HOMO as the S p r Fedr C T band involves with negative values dashed. transitions to the dXz-,z F e r orbital as well as the redox-active d,z
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shown in Figure 2, where the wave function contour for *HOMO has been superimposed onto the Pc structure. newt or^^^^^^ has shown that TDA is proportional to y ~ where ~ , y~ is the ligand covalency in the redox active wave function. The ratio of the Cys/His ligand coefficients in *HOMO indicates that the anisotropic covalency enhances ET through the remote path by a factor of 1500 over the adjacent path, making this a very effective ET route.'* Thus, the unusual electronic structure of the blue copper active site makes a significant contribution to the ET reactivity of this protein. The Rd active &el9 is situated at the surface of the protein and consists of an iron atom tetrahedrally coordinated by the S, atoms of four Cys residues, resulting in an effective active-site symmetry of D2d. The extremely fast ese rate for Rd, estimated4 to be lo9 M-1 s-l, indicates that ET is nearly adiabatic and likely diffusion controlled; however, the path of E T is not known. From Mossbauer spectroscopy,20the r symmetry Fe d,r orbital is known to be the HOMO in reduced Rd. In strict D2dsymmetry, no S(Cys)pn mixing is allowed in the d,2 orbital; however, distortion from ideal D2d geometry in the protein gives rise to some r mixing as evidenced in the C T spectrum of Rd (Figure 1B). In contrast to thevery intense S(Cys)pn+ Cu2+transitions observed for Pc, the S(Cys)pa F e r transitions in Rd are very weak, nearly 2 orders of magnitude weaker than the S(Cys)pu Feu C T bands. The weak S(Cys)pr Fen transitions reflect a limited amount of FeS(Cys) covalency in the HOMO, which will profoundly affect the ET kinetics in this protein. The extent of S(Cys)pr covalency in the HOMO can be estimated from the magnetic circular dichroism (MCD) and electronic absorption spectra2'of [Et4N][Fe(SR)4] (R = 2,3,5,6(CH3)&6H), an iron tetrathiolate model complex that exhibits band energies and intensities similar to those of Rd. From MCD data of the model complex, the spin-orbit coupling constant for the degenerate u Fe3+dXZand d,z orbitals is 65% of the free ion
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orbital. The lack of significant metal-Cg r-bonding in Rd relative to Pc is attributed to the longer F e S length (2.30 A19) and contracted metal orbitals. Employing the relationship between y~ and T D Aand Newton's17 parameters for Fe(H20)62+13+, one can estimate TDAfor a direct outer-sphere ET mechanism in Rd via overlap with the Cys sulfurs. Using (TDA)H~O = 25 cm-l, ( Y L ) H ~ O= 0.06, and ( Y L ) s ( c ~ ~=) 0.10, T D A for ET through the Cys sulfurs in Rd is estimated to be 70 cm-I. A small F e S bond distortion,
